p-Lapachone(p-Lap)(3,4-dihydro-2,2-dimethyl-2H-naphthol[1,2-b]pyran-5,6-dione) is an experimental anti-cancer drug originally obtained from Lapacho trees. P-Lap causes cell death in a variety of human cancer cells. Phase I and II clinical trials to investigate the safety and effectiveness of 3-lap used alone or in combination with other chemotherapy drugs are underway elsewhere. These clinical trials are based on the hypothesis that p-lap kills cancer cells by activating cell cycle checkpoints. On the other hand, our hypothesis is that bioactivation of p-lap through mediation of NAD(P)H:quinone oxidoreductase (NQ01) induces cascade of molecular changes in cells leading to cell death. Therefore, the cytotoxicity of p-lap is closely related to the activity of cellular NQO1 level. Importantly, NQ01 activity in many human tumors is as much as 50 times greater than that in adjacent normal tissues indicating that p-lap may cause greater damage in tumors relative to normal tissues. Moreover, ionizing radiation (IR) causes a long-lasting elevation of NQO1 activity in cancer cells and, thus, IR and p-lap synergistically react in killing cancer cells. We have recently discovered that hyperthermia at clinically achievable temperatures, e.g. <42oC, also causes a long-lasting elevation of NQO1 activity in cancer cells, thereby potentiating the response of cancer cells to p-lap. We hypothesize that established tumor treatment regimens such as radiotherapy and hyperthermia can potentiate the p-lap cytotoxicity towards tumors by increasing NQO1 activity in the tumor cells. The overall aim of the present proposal is to develop effective regimens to selectively enhance the tumor response to p-lap by elevating NQO1 activity in the tumor utilizing radiotherapy and hyperthermia. Specific aims are (1) Determine the effect of IR on the NQO1 level and p-lap sensitivity of cancer cells, (2) Determine the effect of hyperthermia alone or in combination with IR on the NQ01 level and p-lap sensitivity of cancer cells, (3) Elucidate the response of tumors in vivo to P-lap with IR and hyperthermia individually and combined and (4) Delineate cellular and molecular mechanisms of IR- and hyperthermia-induced elevation of NQO1.